Smooth muscle and nonmuscle myosin II are regulated by phosphorylation of the 20 kDa regulatory light chain (RLC) located in the neck region. This region contains a single alpha-helical segment of the myosin heavy chain and the RLC and the essential light chain (ELC). There are three vertebrate genes encoding for nonmuscle myosin IIs, termed IIA and IIB. Platelets express only the IIA isoform, whereas brain expressed predominantly the IIB isoform. Most other cells express nearly equal ratios of IIA and IIB where they are differentially localized. Previous work in our lab has revealed that nonmuscle myosin IIA and myosin IIB have differeing enzymatic and motile properties. In order to better understand the basis for this difference, we have engineered myosin IIA and myosin IIB S-1-like fragments for baculoviral expression. We have fully characterized the transient enzyme kinetics of these two molecules in the presence and absence of actin. Both are characterized by generally slow kinetics (compared to other myosin II isoforms), and each are rate-limted by phosphate release in common with all other myosin II isoforms studied to date. Both myosin IIA and IIB are unusual in that actin does not have a great effect on its ADP affinity and vice versa. Myosin IIB is very unusual in that its ADP release from actomyosin is only three times slower than the rate of phosphate release. Simulations show that the duty ratio (fraction of kinetic cycle spent in high actin affinity AM.ADP or AM states) is 0.2. This is an intermediate value between the usual 0.02-0.05 found for all other myosin II isoforms and the value of 0.6-0.8 found for processive myosins such as myosin V or myosin VI. This implies that the function of myosin IIB in cells may be to hold or maintain tension. We have begun to test this hypothesis using optical trapping nanometry. Preliminary characterization of nonmuscle myosin IIC indicates that it is also a very slow myosin in terms of its rate of actin filament sliding and its steady-state actin-activated MgATPase rate.